Graves' disease is an example of an antibody-mediated inflammatory auto-immune disorder that is clinically manifested as an expansion of the orbital contents and associated severe inflammation of the connective tissue and extra-ocular muscles. Graves' disease is caused by a hyper-functioning, diffuse, hyperplastic thyroid goiter, often accompanied by infiltrative ophthalmopathy and infiltrative dermopathy. Graves' disease is present in 1.5% to 2% of women in the United States, but is only one-tenth as common among men, and is rare in children. Familial predisposition has been noted frequently. A well-defined relationship also exists between Graves' disease and other auto-immune diseases, such as pernicious anemia and rheumatoid arthritis, which occur with greater than normal frequency in patients with Graves' disease. With TAO, characteristic tissue remodeling occurs in the orbital area, including lymphocyte infiltration, hyaluronan accumulation, and inflammation.
Like many auto-immune disorders, Graves' disease and rheumatoid arthritis are difficult to manage clinically because currently available treatments are either ineffective or have significant side effects, due largely to the lack of specificity of the treatment. The absence of any satisfactory therapies is also directly attributable to the current poor understanding of the fundamental biochemical pathways and mechanisms underlying the disease process. Left untreated, subtle changes in cell signalling and molecular immunological mechanisms associated with such disorders result in profound physical defects. Rheumatoid arthritis is painful and the chronic condition can be almost totally debilitating. In TAO, the orbital space is constrained by bone and thus small increases in the volume of soft tissue will cause the anterior displacement of the eye (proptosis). Proptosis occurs in TAO, where the endomysial connective tissue and fat/connective tissue in the orbit are infiltrated with immunocompetent cells such as lymphocytes, macrophages and mast cells. (Smith, T. J., Bahn, R. S. and Gorman, C. A.: Connective tissue, glycosaminoglycans and diseases of the thyroid. Endocrine Rev. 10:366-391, 1989). The inflammatory reaction is sometimes intense. A major component of the tissue remodeling seen in the orbit in TAO relates to the accumulation of the non-sulfated glycosaminoglycan, hyaluronan. Hyaluronan possesses a set of rheological properties that render the molecule extraordinarily hydrophilic (Smith, T. J., Bahn, R. S. and Gorman, C. A.: Connective tissue, glycosaminoglycans and diseases of the thyroid. Endocrine Rev. 10:366-391, 1989). Thus, even a small increase in the hyaluronan content of a tissue could increase its volume dramatically. In the setting of the bony orbit, such an increase could yield catastrophic consequences to the integrity of soft tissue structures, innervation and vascularity.
Debate exists as to whether the primary focus of the pathogenic process in TAO is directed at the extraocular musculature or the connective/adipose tissue in the orbit. In many cases, enlargement of the extraocular musculature appears to be more dramatic than that of the fat pad (Smith, T. J., Bahn, R. S. and Gorman, C. A.: Connective tissue, glycosaminoglycans and diseases of the thyroid. Endocrine Rev. 10:366-391, 1989). The work of Smelser provided strong evidence that TAO is a fibroblast-related disease process (Smelser, G. K.: A comparative study of experimental and clinical exophthalmos. Am. J. Ophthalmol. 20:1189-1203, 1937). This group described the early disease as similar edematous changes occurring in both muscle and fat interstitium with minimal muscle degeneration. Subsequent light and transmission electron microscopic studies have confirmed the preservation of the motor elements of the muscles until very late in the disease process. The endomysium, rich with fibroblasts, accumulates hyaluronan, accounting for the volume changes observed in muscle.
Medical researchers are increasingly aware of the complexity with which connective tissue is regulated and of the diverse influences that elements of this tissue exert on neighboring cell types. Fibroblasts vary with regard to anatomic region of origin and synthesize numerous small regulatory molecules including cytokines and growth factors (Fries, K. M., Blieden, T., Looney, R. J., Sempowski, G. D., Slvera, M. R., Willis, R. A. and Phipps, R. P.: Evidence of fibroblast heterogeneity and the role of fibroblast populations in fibrosis. Clin. Immunol. Immunopath. 72:283-292, 1994). As described in detail herein, depending on the anatomic origin, fibroblasts are increasingly seen as gatekeeper cells for a variety of mechanisms, including specifically immunologic mechanisms that regulate cell signalling and cell activation. For example, the biosynthetic repertoires of orbital fibroblasts are vastly different from those found in other anatomic locations, their responses to hormones and growth factors and the extracellular matrix that they lay down are also different. (Smith, T. J., Aftab, A., Hogg, M. G. and Higgins, P. J.: Interferon-γ is an inducer of plasminogen activator inhibitor type 1 in human orbital fibroblasts. Am. J. Physiol. 263:C24-C29, 1992; Smith, T. J., Bahn, R. S., Gorman, C. A. and Cheavens, M.: Stimulation of glycosaminoglycan accumulation by interferon gamma in cultured human retroocular fibroblasts. J. Clin. Endocrinol. Metab. 72:1169-1171, 1991; Smith, T. J.: Dexamethasone regulation of glycosaminoglycan synthesis in cultured human skin fibroblasts: similar effects of glucocorticoid and thyroid hormones. J. Clin. Invest. 74:2157-2163, 1984; Smith, T. J., Bahn, R. S. and Gorman, C. A.: Hormonal regulation of hyaluronate synthesis in cultured human fibroblasts: evidence for differences between retroocular and dermal fibroblasts. J. Clin. Endocrinol. Metab. 69:1019-1023, 1989). Controversy also exists with regard to the phenotypes of lymphocytes predominating in the orbit of patients with TAO (McLachlan, S. M., Prummel, M. F. and Rapoport, B.: Cell-mediated or humoral immunity in Graves' ophthalmopathy? Profiles of T-cell cytokines amplified by polymerase chain reaction from orbital tissue. J. Clin. Endocrinol. Metab. 78:1070-1074, 1994; Jaume, J. C., Portolano, S., Prummel, M. F., McLachlan, S. M. and Rapoport, B., Molecular cloning and characterization of genes for antibodies generated by orbital tissue-infiltrating B-cells in Graves' ophthalmopathy. J. Clin. Endocrinol. Metab. 78:348-352, 1994; De Carli, M., D'Elios, M. M., Mariotti, S., Marcocci, C., Pinchera, A., Ricci, M., Romagnani, S. and Del Prete, G.: Cytolytic T cells with Th1-like cytokine profile predominate in retroorbital lymphocyte infiltrates of Graves' ophthalmopathy. J. Clin. Endocrinol. Metab. 77:1120-1124, 1993; Grubeck-Loebenstein, B., Trieb, K., Sztankay, A, Holter, W., Anderi, H. and Wick, G.: Retrobulbar T cells from patients with Graves' ophthalmopathy are CD8+ and specifically recognize autologous fibroblasts. J. Clin. Invest. 93:2738-2743, 1994).
The selective targeting of orbital connective tissue for activation in TAO associated with Graves' disease is not currently understood. A central question is why activated T and B lymphocytes are trafficked to the orbit. Interest has centered on the successful cloning of the thyrotropin or Thyroid Stimulating Hormone Receptor (TSHr) because patients with Graves' disease typically express high levels of antibodies against the TSHr receptor, which can be either stimulatory or inhibitory. Stimulatory anti-TSHr antibodies activate the receptor and yield increased cAMP levels, thyrocyte proliferation and thyroid hormone overproduction. Inhibitory TSHr antibodies attenuate thyroid gland activity. Studies examining the role of stimulatory and inhibitory anti-TSHr antibodies on the metabolism of cultured fibroblasts have been inconsistent. Rotella, C. M., Zonefrati, R., Toccafondi, R., Valente, W. A., and Kohn, L. D.: Ability of monoclonal antibodies to the thyrotropin receptor to increase collagen synthesis in human fibroblasts: an assay which appears to measure exophthalmogenic immunoglobulins in Graves' sera. J. Clin. Endocrinol. Metab. 62:357-367, 1986; Cheung, H. S., Nicoloff, J. T., Kamiel, M. B., Spolter, L. and Nimni, M. E.: Stimulation of fibroblasts biosynthetic activity by serum of patients with pretibal myxedema. J. Invest. Dermatol. 71:12-17, 1978.
TSHr is also implicated in the glandular component of Graves' disease by virtue of its activation by Thyroid Stimulating Immunoglobulin (TSI) resulting in hyperthyroidism. Recently, preliminary evidence was introduced suggesting that TSIs are heterogeneous (Drexhage, H. A.: Autoimmunity and thyroid growth. Eur. J. Endocrinol. 135:39-45, 1996). Moreover, different sub-classes of TSI might activate distinct signaling pathways in the thyroid. Until now, little insight existed concerning the actions of TSI on orbital fibroblasts. Thus, no connection had been identified between the presence of these immunoglobulins and the pathogenesis of TAO. A number of investigators have speculated that the TSHr might represent an auto-antigen relevant to TAO if anatomically restricted expression, shared by the orbit and the thyroid, could be established. Indeed, mRNA encoding the TSHr has been extracted from the orbits of patients with severe TAO as well as from normal orbital tissue (Feliciello, A., Porcellini, A., Ciullo, I., Bonavolonta, G., Avvedimento, E. V. and Fenzi, G., Expression of thyrotropin-receptor mRNA in healthy and Graves' disease retro-orbital tissue. Lancet 342:337-338, 1993). Moreover, TSHr mRNA has been detected in orbital fibroblasts by PCR amplification (Heufelder, A. E., Dutton, C. M., Sarkar, G., Donovan, K. A. and Bahn, R. S.: Detection of TSH receptor RNA in cultured fibroblasts from patients with Graves' ophthalmopathy and pretibial dermopathy. Thyroid 3:297-300, 1993).
Pro-inflammatory cytokines also have been implicated in the pathogenesis of TAO and orbital fibroblasts exhibit exaggerated responses to pro-inflammatory cytokines. A recent report suggested the presence of immunoreactive IL-1α in orbital connective tissue from patients with severe TAO (Heufelder, A. E. and Bahn, R. S.: Detection and localization of cytokine immunoreactivity in retro-ocular connective tissue in Graves' ophthalmopathy. European J. Invest. 23:10-17, 1993. 22. Dinarello, C. A.: Biologic basis for interleukin-1 in disease. Blood 87:2095-2147, 1996). IL-1 is a family of two cytokines designated IL-1α and IL-1β, each encoded by a separate gene but with substantial overlap in their biological actions (Dinarello, C. A.: Biologic basis for interleukin-1 in disease. Blood 87:2095-2147, 1996). IL-1α is primarily an intracellular molecule while IL-1β is exported to the outside of the cell expressing it. It would appear that these IL-1 proteins share common receptors. Bahn has also implicated leukoregulin, a T lymphocyte-derived cytokine, in the pathogenesis of TAO (Bahn, R. S.: Cytokines in thyroid eye disease. Thyroid 8:415-418, 1998). Leukoregulin is a 50 kDa cytokine that is expressed by activated T lymphocytes (Mauviel, A., Redini, F., Hartmann, D. J., Pujol, J.-P. and Evans, C. H.: Modulation of human dermal fibroblast extracellular matrix metabolism by the lymphokine leukoregulin. J. Cell Biol. 113:1455-1462, 1991) and acts through NF-KB to up-regulate inflammatory and extracellular matrix-encoding genes.
Thy-1 is a 25-35 kDa glycoprotein expressed by a wide variety of cells, although whose function as a receptor is uncertain. Thy-1+ and Thy-1− are heterogeneously present in orbital fibroblasts. Approximately 50 to 60 percent of orbital fibroblasts in both TAO and normal orbital connective tissue express the Thy-1 receptor. Although the presence of the Thy-1 receptor is heterogeneous on orbital fibroblasts, Thy-1+ and Thy-1− cells exhibit distinct and different phenotypes. Each phenotype may be characterized by expression of PGHS-2 upon exposure to IL-1 beta and the production of large amounts of PGE2, although Thy-1+ cells produce more PGE2 than Thy-1− fibroblasts. Thy-1− cells are implicated in exaggerated foreign antigen presenting activity and have a greater potential for activating macrophages and lymphocytes. Therefore, while Thy-1+ and Thy-1− fibroblasts exhibit different phenotypes and appear to participate differently in fundamental immune mechanisms and cell trafficking, the distinctions are not well-characterized.
IL-16 is a CD4-specific chemoattractant molecule not assigned to any chemokine family because the requisite cysteine signature residues for such designation are absent (Center, D. M., Kornfeld, H. and Cruikshank, W. W.: interleukin 16 and its function as a CD4 ligand. Immunol. Today 17:476-481, 1996). IL-16 was originally found to be expressed by activated CD8+ lymphocytes but subsequently has been found to be produced by mast cells, bronchial epithelium, and CD4+ lymphocytes. It has been cloned and found to be regulated by several factors in a cell-type-specific pattern. IL-16 is a CD4 ligand and when bound, leads to lymphocyte activation and IL-2 receptor up-regulation. It has been implicated in the pathogenesis of asthma and human auto-immune diseases such as rheumatoid arthritis and lupus. IL-16 was detected in synovial fluid and soft tissues of diseased joints (Franz, J. K., Kolb, S. A., Hummel, K. M., Lahrtz, F., Neidhart, M., Aicher, W. K., Pap, T., Gay, R. E., Fontana, A. and Gay, S.: Interleukin-16, produced by synovial fibroblasts, mediates chemoattraction of CD4+ T lymphocytes in rheumatoid arthritis. Eur. J. Immunol. 28:2661-1998).
In addition to TAO, several other connective tissue disorders have been shown to have an antibody-mediated auto-immune component. For example, vitiligo, a depigmenting order of the skin, is caused by the destruction of melanocytes. Although the cause is still unknown, prominent theories explaining the mechanism of melanocyte destruction are auto-immune, autocytotoxicm and neural hypotheses. (S. O. Kovacs, Vitiligo, 38 J. Am. Acad. Dermatol. 647 (1998)). Pemphigus vulgaris is another antibody-mediated auto-immune disease affecting the skin. Antibodies against desmosomal adhesion molecules (Dsg3) are thought to be a major factor in the pathogenesis of the disease. Autoreactive T-cells, which recognize epitopes of Dsg3 in PV patients, preferentially produce TH2 cytokines such as IL-4 and IL-10. (M. Hertl and R. Riechers, Analysis of the T cells that are Potentially Involved in Autoantibody Production in Pemphigus Vulgaris, 26 J. Dermatol. 748 (1999).
Polyglandular auto-immune syndrome is used to describe the dysfunction of two or more endocrine glands occurring in association with circulating antibodies directed against the affected glands. The auto-immune nature of the syndrome is caused by the presence of lymphocytic infiltration of the affected gland, organ-specific autoantibodies, cellular immune defects, and an association with the immune response genes. The principal endocrine components include adrenal insufficiency, auto-immune thyroid disease, insulin-dependent diabetes mellitus, and premature gonadal failure. (M. Leshin, Polyglandular Autoimmune Syndromes, 290 Am. J. Med. Sci. 77 (1985); W. J. Riley, Autoimmune Polyglandular Syndromes, 38 Horm. Res. 9 (Suppl. 2) (1992)).
Type 1 diabetes, a disease affecting several million every year, is caused by auto-immune destruction of the B-cells in the pancreatic islets of Langerhans which produce insulin. Although it is required to be present, a certain genetic phenotype does not appear sufficient on its own to trigger the disease. Many believe that infectious agents are important environmental factors which help trigger T-cell activation. (P. Luppi and M. Trucco, Immunological Models of Type 1 Diabetes, 52 Horm. Res. 1 (1999)).
Systemic lupus erythematosus (SLE) is a non-organ specific auto-immune disease. The primary autoantigen responsible for the disease is not currently known. Although it was previously believed that DNA-AntiDNA complexes mediated the disease, it is now thought that nucleosomes and complement factor CLq play roles in the pathogenesis. (S. O. McLigeyo, Pathogenesis of Lupus Nephritis: a Review, 75 East Afr. Med. J, 628 (1998)).
Nephritis is a common complication of SLE. In this immune-mediated disease, nephritogenic autoantibodies which localize to the kidney are accompanied by activated macrophages and T cells as a result of enhanced, abnormal production of macrophage growth factors and cytokines. (M. H. Foster and V. R. Kelley, Lupus nephritis: Update on Pathogenesis and Disease Mechanisms, 19 Semin. Nephrol. 173 (1999)).
Rheumatoid arthritis is a chronic multisystem auto-immune inflammatory disease where an unknown autoantigen is presented to CD4+ T cells. The immune response primarily occurs in the synovial tissue and fluid of the joints. (Thomas R. MacDonald et al., Dendritic Cells and the Pathogenesis of Rheumatoid Arthritis, 66 J. Leukoc. Biol. 286 (1999)).
Although there are several treatments for the overt physiologically manifested symptoms of these disorders, an acute need exists for therapies which are directed toward altering the underlying mechanisms and biochemical pathways that cause these disorders. Moreover, techniques are needed to diagnose such disorders, as well as to identify specific mechanisms that are present in patients such that drug therapy, or surgical intervention, can be specifically selected in accord with the underlying pathologies of the disease.